The long-term goal of the proposed research is to understand how cardiac mitochondria initiate apoptosis and how moderate alcohol consumption may act as a therapeutic modality to block or to retard apoptotic signaling associated with the progression of heart disease. Moderate alcohol consumption is linked to a lower risk of coronary artery disease and a decreased risk of ischemic injury. Programmed cell death now appears to play a quantitatively more important role in the loss of myocyte viability than previously recognized. Since both ischemic heart disease and the initiation of apoptosis are linked to changes in intracellular calcium metabolism, we propose to examine the relation of mitochondrial calcium to apoptotic events and how these pathways are modulated by brief but chronic exposure of cardiac myocytes to small concentrations of alcohol. Effects of alcohol on the cardiac cytoskeleton will be monitored to assure absence of toxicity. We are able to visualize directly and quantitate cellular and mitochondrial changes in a longitudinal fashion by using palmitate to induce apoptosis in neonatal rat cardiomyocytes in culture. By using this approach, we propose to elucidate the role of mitochondrial calcium in this process and whether alcohol prevents progression to the mitochondrial permeability transition with a loss in membrane potential and subsequent activation of cell death signaling. By using a combination of biochemical and microscopic tools, the ability of alcohol to attenuate mitochondrial calcium overload leading to the permeability transition and cytochrome c release will be studied. We propose to test whether the anti- oxidant properties of alcoholic beverages or whether direct effects of alcohol block the release of mitochondrially-generated free radicals and the subsequent loss of the mitochondrial membrane potential. Finally, we will look at the role of fatty acid metabolism and ceramide formation on signal transduction pathways that may be individually altered by exposure of cardiac myocytes to low levels of alcohol.